The objectives of the proposed research are to understand the mechanisms for the generation and emergence of variants during chronic infection with hepadnaviruses. Variants are thought to be important in the pathogenesis or persistence of HBV infections in humans, and are known to be responsible for the development of drug resistance during antiviral therapy and for infection in vaccinated individuals and liver transplant recipients. However, because chronic infection is due to persistent infection at the cellular level, and the liver is a relatively quiescent tissue, it is not clear how variants that are generated during a chronic infection emerge as the dominant species of virus. The research we propose is designed to clarify the mechanisms for emergence of virus strains. Understanding the processes that result in evolution of the virus population in a chronically infected liver requires gaining new fundamental information about the nature of chronic infections; i.e. about how the progeny of the infecting viruses and variants are distributed in the liver, how much viral and cellular turnover occurs during chronic infection, and how virus lineages are segregated in different populations of hepatocytes where selection of individual variants can occur. Because these questions cannot be addressed experimentally in human HBV infections, we will use the duck hepatitis B virus (DHBV) model. DHBV, a member of the hepadnavirus family, closely resembles HBV in virus structure, genome replication, persistence, and tissue tropism. DHBV is the only member of this family of viruses that can be genetically manipulated in vitro and tested conveniently in animal infections. The five specific aims are (1) to measure the spontaneous mutation frequency in a single round of transcription and reverse transcription of the DHBV genome, (2) to determine the nature of mixed infections of the liver at the cellular level, particularly the frequency of doubly and singly infected hepatocytes, cell-to-cell variation in covalently closed circular DNA (cccDNA) copy number, and the effects of liver growth and regeneration on these properties, (3) to measure the dynamic state of in vivo chronic infections, (4) to describe the process of replacement of a cytopathic strain by a noncytopathic reverant in vivo, and (5) to determine the spatial distribution of virus strains in mixed infections.